Leporipox-based vector vaccines

ABSTRACT

The present invention is directed to the use of a live, recombinant leporipox virus comprising exogenous DNA, which is operably linked to at least one expression control element and which is incorporated in a non-essential region of the virus genome, in the manufacture of a vector vaccine for the treatment and/or prophylaxis of infectious diseases in non-lepori species. The invention furthermore relates to a live, recombinant leporipox virus comprising exogenous DNA operably linked to at least one expression control element and incorporated in a non-essential region of the virus genome characterized in that said exogenous DNA encodes at least one antigen of a non-lepori pathogen. Due to its restricted host-range the recombinant leporipox virus is non-pathogenic in non-susceptible hosts such as non-lepori vertebrates. Vaccination with said recombinant leporipox virus induced an antigen or immunogenic response in the vaccinated non-lepori host even though productive replication of the virus was not observed in the host.

[0001] The present invention relates to the use of a leporipox virusvector vaccine in non-susceptible host species and live, recombinantleporipox viruses.

[0002] Vector vaccines based on orthopox and avipox viruses and theirpotential as recombinant viral vectors in vaccination have beendescribed. U.S. Pat. No. 5,759,841 describes a recombinant vacciniavirus which contain morbillivirus DNA coding for at least oneglycoprotein, and a promoter for expression of the DNA, In anon-essential region of the vaccinia virus genome. The recombinantvaccinia virus can be used in vaccines for inducing an immune responseto morbillivirus in dogs. The recombinant vaccinia vector virus howeveris permissive in a great number of different species including humanshence the described vaccinia vector virus has the potential risk ofcausing a runaway infection in the vaccinated host or of transmissionfrom vaccinated to unvaccinated hosts.

[0003] WO 9527780 describes a recombinant avipox virus, which by virtueof its restricted host-range has attenuated virulence in a non-avianhost The recombinant avipox viruses contain exogenous DNA in anon-essential region of the virus genome, whereby the exogenous DNAencodes at least one Canine Distemper virus (CDV) antigen, or measlesvirus (MV) M or N antigen. These viruses can be used to induce anantigenic or immunologic response in canines and other carnivores aswell as in humans. The recombinant avipox vector viruses are restrictedto their natural host and vaccination of non-avian species with saidvector viruses results in expression of the exogenous antigen withoutproductive replication of the virus. However the level of expression ofthe exogenous antigen remains low after vaccination with the avipoxvirus vector. Hence there is a need for improved expression levels ofthe exogenous antigen. Furthermore immunization with avipox virus vectordoes not always provide sufficient neutralizing antibodies against theexogenous antigen. Vaccination of cats with a canary pox-based FeLVvector vaccine did not lead to the production of neutralizing anti-FeLVantibodies (J. Tartaglia et al., 1993, J. Virol. 67, p. 2370-2375).Newborn kittens are especially susceptive to FeLV infection. Since theydo not have a matured immune system in the first weeks after birth,newborn kittens have to rely on the maternally derived antibodies forprotection against FeLV infection. If vaccination did not provide themother with neutralizing antibodies, the kittens will not be protectedagainst FeLV and they will succumb to the infection.

[0004] Surprisingly it was found that a live recombinant leporipox viruscomprising exogenous DNA encoding at least one antigen could be used toinduce an antigenic or immunogenic response in a host which is normallynot susceptible to productive infection of leporipox virus i.e. theleporipox virus is not able to replicate in said host after replication.Productive infection of leporipox viruses is restricted to leporispecies only. Consequently infection of non-lepori species with aleporipox virus will not lead to replication of the leporipox virus. Itwas therefore surprising to find out that a live recombinant leporipoxvirus was capable of infecting a non-susceptible host and expressingsaid antigen in the absence of productive replication of the recombinantvirus in said host, as evidenced by the fact that shedding of the virusvector to any other contact animal does not occur. More surprisingly,infection of a non-lepori host with said leporipox virus vector resultedin high expression levels of the antigen encoded by the exogenous DNAeven though productive replication of the virus in said host was notobserved. Growth of the viral vector in vitro does occur in somemammalian cell lines. Furthermore, due to the absence of productivereplication of the leporipox virus vector in a such a non-susceptiblehost, the leporipox virus will be non-pathogenic in the non-leporispecies, which makes these virus vectors even more suitable forvaccination.

[0005] Vaccination with a recombinant myxoma virus comprising exogenousDNA have been described in FR-A-2736358. The recombinant myxoma viruswas used to vaccinate rabbits against myxomatosis and infectiousdiseases caused by other rabbit pathogens. Nowhere does FR-A-2736358suggest the use of a live, recombinant myxoma virus as viral vector toinduce an antigenic or immunogenic response in non-susceptible species,more particular non-lepori species.

[0006] Hence the present invention pertains to the use of a live,recombinant leporipox virus comprising exogenous DNA, which is operablylinked to at least one expression control element and which isincorporated in a non-essential region of the virus genome, in themanufacture of a vector vaccine for the treatment and/or prophylaxis ofinfectious diseases in non-lepori species. Preferably a live,recombinant myxoma virus comprising exogenous DNA, which is operablylinked to at least one expression control element and which isincorporated in a non-essential region of the virus genome, is used inthe manufacture of a vector vaccine for the treatment and/or prophylaxisof infectious diseases in non-lepori species. More specifically theinvention concerns the use of said live, recombinant leporipox virus inthe manufacture of a vector vaccine for the treatment and/or prophylaxisof infectious diseases in avian-, feline-, canine-, porcine-, ovine-,bovine-, equine-, and human species. Preferably the live recombinantleporipox virus according to the invention is used to manufacture avector vaccine for the treatment of infectious diseases in canine- andfeline species.

[0007] The invention furthermore provides for a live recombinantleporipox virus comprising exogenous DNA operably linked to at least oneexpression control element, said exogenous DNA encoding at least oneantigen of a pathogen that produces an infectious disease innon-leporidae. More specifically the exogenous DNA preferably encodes atleast an antigen of a pathogen that causes an infectious disease inhuman-, bovine-, avian-, feline-, canine-, porcine-, equine- or ovinespecies. Preferably the exogenous DNA encodes an antigen of a feline- orcanine pathogen. According to the invention the pathogen can be ofviral-, bacterial or parasitic origin, depending on the disease againstwhich the subject has to be vaccinated. If the pathogen has an RNAgenome, the antigen of interest may be encoded by cDNA corresponding tothe gene. The exogenous DNA may encode two or more antigens, which canbe derived from the same pathogen or from different pathogens.

[0008] Suitable exogenous DNA for use in a recombinant leporipox viruspreferably encodes viral glycoproteins, viral envelope proteins, viralmatrix proteins, bacterial outer membrane proteins, bacterialenterotoxins, bacterial fimbriae or parasitic proteins. The exogenousDNA more specifically encodes the Feline Leukaemia virus (FeLV) envelopeprotein (Stewart et al. (1986) J. Virol. 58 pp. 825-834) or matrixprotein (Donahue et al., 1988, J. Virol. 62, p. 722-731), feline- orsheep chlamidia major outer membrane protein (GenBank Accession No.'sCPFPNMOMP and CHTMOMPX, respectively), feline panleukopenia virus (FPV)VP2 protein (Carlson, J. et al., 1985, J. Virol. 55, p. 574-582), felinecalicivirus capsid protein (M. J. Carter et al. 1992, J. Arch. Virol.122, p. 223-235), feline immunodeficiency virus (FIV) Gag, Pol, Rev, Tator Vif proteins (R. I. Talbot et al. 1989, Proc. Natl. Acad. Sci. USA86, p. 5743-5747; T. R. Philips et al. 1990, J. Virol. 64, p. 4605-4613;K. M Lockridge, et al. 1999, J. Virol. 261, p. 25-30), feline infectiousperitonitis virus (FIPV) membrane-, nucleocapsid- or spike protein (R.J. de Groot et al. 1987, J. Gen. Virol. 68, p. 2639-2646; H. Vennema, etal. 1991, Virology, 181, p. 327-335), canine distemper virus Env, HA,fusion- or nucleocapsid protein (M. Sidhu, et al. 1993, Virology 193, p.66-72; U. Gassen, et al. 2000, J. Virol. 74, p. 10737-10744), canineparvovirus VP2 protein (Reed, P. et al., 1988, J. Virol. 62, p.266-276), rabies virus glycoprotein G (T. J. Wiktor, et al. 1984, Proc.Natl. Acad. Sci. USA 81, p. 7194-7198), canine corona virus spikeprotein (B. Horsburgh, et al. 2000, J. Gen,. Virol. 73, p. 2849-2862).In addition to genes encoding immunogenic proteins from non-leporipathogens, the exogenous DNA may also comprise genes encoding cytokinessuch as for example INFγ (GenBank Acc. No. D30619), IL-1β (GenBank Acc.No. M92060), IL-2/15 (GenBank Acc. No. AF054601), IL4, IL-5 (GenBankAcc. No. AF025436), IL-6, IL-12 (GenBank Acc. No. U83184 and U83185),IL-16 (GenBank Acc. No. AF003701) or IL-18 (GenBank Acc. No. ABO46211),or chemotactic cytokines such as the α-chemokines IL-8 (GenBank Acc. No.XM003501), GROα, GROβ, NAP-2, PF4, IP10, CTAP-III, β-TG and theβ-chemokines MCP-1 (GenBank Acc. No. NM002982), MIP-1α, MIP-1β, RANTES(GenBank Acc. No. XM012656), MCP-2 (GenBank Acc. No. AJ251190), MCP-3(GenBank Acc. No. NM 006273), MCP-4 (GenBank Acc. No. AJ251191).Preferably the genes encoding suitable cytokines according to theinvention are derived from the same species the vaccine will beadministered to.

[0009] The exogenous DNA is operably linked to at least one expressioncontrol element, which will control and regulate the expression of saidexogenous DNA. In a preferred embodiment each gene present in theexogenous DNA is controlled by a separate and distinct expressioncontrol element. Expression control elements are known in the art andinclude promoters. Suitable promoters for expression of the exogenousDNA according to the invention are viral or synthetic promoters, whichare able to modulate expression in the cytoplasm. Promoters useful inthe present invention are poxvirus promoters, preferably a vacciniapromoter (see DE-A19627193; Mackett et al., “DNA Cloning Volume III”,ed. D. M. Glover, 1985, IRL Press Ltd.). Preferred promoters accordingto the invention are synthetic promoters, more preferably syntheticearly- or early/late promoters. Synthetic vaccinia virus early/latepromoters are described in Chakrabati et al., BioTechniques 23, vol. 6,pp. 1094-1097, 1997. The promoters can be synthesized by using standardtechniques in the art, such as for example described in Chakrabafi etal., 1997 supra.

[0010] Suitable leporipox viruses that can be used according to theinvention include but are not limited to myxoma viruses or Shope Fibromaviruses. Suitable myxoma virus strains include Lausanne strain (fromATCC), SG33 (Mainil, M. D. et al. 2000, J. Comp. Pathol. 122, p.115-122). Borghi and Boerlage (Fenner & Fantini, “Biological control ofVertebrate Pests”, CABI publishing 1999, ISBN 0 85199 323 0 andreferences therein). Suitable Shope Fibroma Virus strains includeOriginal A strain (ATCC cat. No. VR-112) and Kasza strain (ATCC cat. No.VR-364). Preferably the live recombinant leporipox virus according tothe invention is derived from a rhyxoma virus. Due to itshost-restriction to lepori species, the leporipox virus is not virulentin a non-lepori host. It is however preferred to use an attenuatedleporipox virus to generate the live recombinant viruses of theinvention. For the purpose of the invention an attenuated leporipoxvirus is defined as a leporipox virus that is capable of productivereplication in its target lepori host without causing disease.Attenuation of Leporipox virus strains can be carried out by serialpassage of the strain or by deletion of one or more virulent genes thatare not essential for viral replication. The complete DNA sequence ofleporipox virus genome, its genomic organization and the localization ofall open reading frames (ORF's) is presented in Cameron et al., Virology264, p. 298-318 (1999). The complete DNA sequence of Shope Fibroma virusgenome, its genomic organization and the localization of all openreading frames (ORF's) is presented in Willer et al., Virology 264, p.319-343, (1999).

[0011] The exogenous DNA according to the invention is preferablyinserted in a non-essential gene region of the leporipox virus genome.More preferably the exogenous DNA is inserted in a non-essential generegion that is involved in the virulence of the leporipox virus.Suitable non-essential gene regions of the myxoma virus genome or ShopeFibroma virus genome are the TK gene encoding Thymidine kinase, the M11LORF, SERP-1, -2 and -3 ORF's and MGF ORF (Cameron et al, 1999, supra;Willer et al, 1999 supra). In a preferred embodiment of the inventionone or more of the non-essential viral genes are deleted followed byinsertion of the exogenous DNA and promoter. Deletion of at least partof the MGF ORF is especially preferred since this ORF encodes avirulence factor and is not essential for growth in vitro or in vivo(Graham et al., Virology 191, pp. 112-124, 1992). Deletion of the MGFORF results in a decreased virulence of the leporipox virus.

[0012] The live recombinant leporipox virus according to the presentinvention can be produced using the in vivo recombination technique thatinvolves insertion by site specific recombination of exogenous DNA intothe leporipox virus genome. This can be accomplished using a methodsimilar to the methods described for production of recombinant vacciniavirus and recombinant fowl pox virus (see U.S. Pat. No. 4,603,112; U.S.Pat. No. 5,093,258; Guo, P. X.; J. Virol. 63: 4189-4198 (1990); Mackettet al., “Construction And Characterization Of Vaccinia VirusRecombinants Expressing Exogenous Genes” in “DNA Cloning Volume III” ed.D. M. Glover, 1985, IRL Press Ltd.). In general, the live, recombinantleporipox virus according to the present invention can be produced usingsite-specific recombination between a parental leporipox genome and aDNA vector carrying the exogenous DNA under control of at least oneexpression control element. Suitable DNA vectors for use insite-specific recombination can be derived from any plasmid thatcomprises a multiple cloning site. The DNA vector comprises theexogenous DNA linked to at least one expression control element andlocated between viral DNA sequences homologous to a region of theleporipox genome into which the exogenous DNA is to be incorporated. Theviral DNA sequences flanking the exogenous DNA are preferably selectedfrom a region that is nonessential for replication of the leporipoxvirus. The DNA vector for recombination with the leporipox genome mayadditionally comprise a gene that codes for a selection marker undercontrol of a pox virus promoter. The additional gene and promoter arealso located between the viral DNA sequences derived from the leporipoxgenome. The DNA vector is transfected into host cells infected with aparental leporipox virus. Suitable host cells are eukaryotic cells whichare permissive for the leporipox virus and which are transfectable bythe DNA vector. Examples of host cells are rabbit kidney cells LLC-RK1and RK13, rabbit lung cells R9ab, rabbit skin cells SF 1 Ep, DRS andRAB-9, rabbit cornea cells SIRC, rabbit carcinoma cells Oc4T/cc, rabbitskin/carcinoma cells CTPS, Vero cells, all available from ATCC.

[0013] Parental leporipox virus suitable for generating the live,recombinant leporipox viruses of the present invention are myxoma virusstrains such as Lausanne strain (from ATCC), SG33 (Mainil, M. D. et al,2000, J. Comp. Pathol. 122, p. 115-122), Borghi and Boerlage (Fenner &Fantini, “Biological control of Vertebrate Pests”, CABI publishing 1999,ISBN 0 85199 323 0 and references therein), and Shope Fibroma Virusstrains including Original A strain (ATCC cat. No. VR-112) and Kaszastrain (ATCC cat. No. VR-364). Preferably myxoma virus strains are usedto produce the live recombinant lepori virus according to the invention.Preferably the parental leporipox virus is an attenuated virus i.e. aleporipox virus that is able to productively replicate in its targetlepori host without causing disease. Attenuation of Leporipox virusstrains can be carried out by serial passage of the strain or bydeletion of one or more virulent genes that are not essential for viralreplication (for complete genomic sequence and localisation of genes seeCameron et al. 1999, supra and Willer et al. 1999, supra).

[0014] The virus is allowed to replicate in the host cell during whichrecombination occur between the leporipox DNA sequences on the DNAvector and the corresponding DNA on the parental leporipox genome. Therecombination results in the insertion of the exogenous DNA linked tothe expression control element(s) into the leporipox genome. Therecombinant leporipox viruses are selected and purified using standardselection or screening methods well known in the art including detectionof the integrated exogenous DNA by hybridization with probes homologousto the exogenous DNA, detection of expression of the selection markerco-integrated with the exogenous DNA, and detection of absence of theexpression product of the deleted leporipox gene into which theexogenous DNA has been incorporated. Insertion of the exogenous DNA inthe recombinant leporipox viral genome can be confirmed by polymerasechain reaction analysis.

[0015] The recombinant leporipox virus vector according to the inventionis especially suitable for use as immunizing agent in non-leporidaebecause expression levels of the antigen can be reached in vivo that aresufficient for immunization of the host. Due to its restrictedhost-range the virus is attenuated in a non-lepori host hence there isno risk of disease caused by the leporipox virus. The host-restrictionwill furthermore prevent the leporipox viruses according to theinvention from spreading among hosts, which are not targeted forvaccination. Thus in a further embodiment the present invention providesfor a pharmaceutical composition, more preferably a vaccine comprising apharmaceutical acceptable carrier and a live recombinant leporipox viruscomprising exogenous DNA operably linked to at least one expressioncontrol element and incorporated in a non-essential region of the virusgenome, said exogenous DNA encoding at least one antigen of a pathogenthat produces an infectious disease in non-leporidale. The vaccineaccording to the invention preferably comprises a pharmaceuticalacceptable carrier and a live recombinant myxoma virus according to thepresent invention expressing at least an immunogenic protein of anon-lepori pathogen. A recombinant leporipox virus according to theinvention expressing two or more immunogenic proteins is specificallysuitable for the manufacture of a multivalent vaccine.

[0016] Vaccine compositions according to the invention can be preparedfollowing standard procedures. The recombinant leporipox virus can begrown on a cell culture for which the virus is permissive such as rabbitkidney cells LLC-RK1 and RK13, rabbit lung cells R9ab, rabbit skin cellsSF 1 Ep, DRS and RAB-9, rabbit cornea cells SIRC, rabbit carcinoma cellsOc4T/cc, rabbit skin/carcinoma cells CTPS, Vero cells, all availablefrom ATCC. The viruses thus grown can be harvested by collecting thetissue cell culture fluids and/or cells. Optionally, during harvestingthe yield of the viruses can be promoted by techniques that improve theliberation of the infective particles from the growth substrate, e.g.sonication and freeze thawing. The live vaccine may be prepared in theform of a suspension or may be lyophilized.

[0017] Pharmaceutical acceptable carriers that are suitable for use in avaccine according to the invention are sterile water, saline, aqueousbuffers such as PBS and the like. In addition the vaccine according tothe invention may comprise other additives such as adjuvants,stabilizers, anti-oxidants and others.

[0018] Suitable stabilizers are for example carbohydrates includingsorbitol, mannitol, starch, sucrose, dextran and glucose, proteins anddegradation products thereof including but not limited to albumin andcasein, protein-containing agents such as bovine serum or skimmed milk,and buffers including but not limited to alkali metal phosphates. Inlyophilized vaccine compositions it is preferable to add one or morestabilizers.

[0019] Suitable adjuvants include but are not limited to aluminumhydroxyde, phosphate or oxide, amphigen, tocophenols, monophosphenyllipid A, muramyl dipeptide, oil emulsions, glucans, carbomers, blockcopolymers, cytokines and saponins such as Quil A. The amount ofadjuvant added depends on the nature of the adjuvant itself. Cytokinessuch as INFγ, IL-12₁₂, IL18 are very suitable for use in a vaccineaccording to the invention.

[0020] Preferably the recombinant leporipox viruses according to theinvention are administered to the non-lepori species via parenteraladministration routes including but not limited to intramusculair,intradermal, or subcutaneous routes. Alternatively, the vaccine can beadministered via non-parenteral administration routes such as oral,spraying, intra-ocular, intranasal or in ovo administration.

[0021] In general the recombinant leporipox virus according to theinvention is administered in an amount that is effective to induceadequate expression levels of the exogenous protein. The dose generallywill depend on the route of administration, the time of administration,as well as age, health and diet of the animal to be vaccinated. Therecombinant leporipox virus can be administered in an amount between 10²and 10¹¹ pfu/dose per subject, preferably between 10⁴ and 10⁹ pfu/doseand more preferably 10⁶ to 10⁷ pfu/dose per subject (pfu is “plagueforming units”).

[0022] The vaccines according to the invention also may be givensimultaneously or concomitantly with other live or inactivated vaccines.These additional vaccines can be administered non-parenteral orparenteral. Preferably the additional vaccines are recommended forparenteral administration.

[0023] The following experiments are illustrative for the invention anddo not limit the invention to the particular embodiments described.

LEGENDS TO THE FIGURES

[0024]FIG. 1: schematic representation of construction of intermediateplasmids pV_(L) and PV_(EL). RHD represents cDNA of rabbit haemorrhagicvirus. ab(5′) and cd(3′) represent the myxoma virus MGF flankingregions. Promoter represents synthetic late or early/late promoter,respectively. “mcs” represents nucleotide sequence comprising multiplecloning sites for introduction of exogenous DNA.

[0025]FIG. 2: the various recombinant DNA plasmids based on pV_(L)pV_(EL) which have been constructed. P represents the synthetic late(pV_(L)) or early/late (pV_(EL)) promoter region; ab(5′) and cd(3′)represent the myxoma virus MGF flanking regions. RHDV Vp60 represent thegene encoding RHDV VP60 protein. FeLV gp85 represents the FelineLeukaemiavirus env gene. FCV Vp60 represents gene encoding felinecalicivirus capsid protein. FPL Vp2 represents feline panleukopeniavirus vp2 gene. CPV VP2 represents canine parvovirus vp2 gene. GFPrepresent gene encoding green fluorescent protein. All plasmids compriseAmp_(r) as selection marker (not shown).

EXAMPLES Example 1 Preparation of intermediate DNA plasmids pV_(L) andpV_(EL)

[0026] The starting plasmid for the procedure was the commerciallyavailable plasmid pCITE 2-b (Novagen inc.) containing a cDNA of rabbithaemorrhagic disease virus (Meyers G., et al. 1991, Virology 184, p.664-676) inserted into the Sall and Hincil sites of the vector. Thisplasmid is referred to as pCITE/RHD

[0027] The first step was the introduction of the MGF flankingsequences. PCR primers myx a and myx b were used to amplify the 5′flanking sequence. myx a: 5′ TTCTCGGAAGTCATAGACGGTATT 3′ (seq id no 1)myx b: 5′ CATGCCAATGGCACATAAGAGAGTTGCGACTAGGTC 3′ (seq id no 2)

[0028] A 2 μl sample of tissue culture grown MR24 (10⁶ pfu ml⁻¹) wasused as template for the PCR reaction, which was carried out using PCRBeads (Pharmacia) following the manufactures instructions. The PCRfragment was cloned using standard laboratory methods as a NcoI/bluntfragment into pCITE/RHD. The pCITE/RHD was first prepared by digestionwith KpnI, followed by “blunting” with T4 DNA polymerase, then digestionwith NcoI. The resulting plasmid was called pCITE/RHDab.

[0029] A second PCR reaction on an identical template preparation usingthe primers myx c: 5′ CGGCTCGAGCTAATTACCATTAAGTAACCCGTTTTACA 3′ (seq idno 3)        XhoI myx d: 5′GCTCTAGATATATCGTGTACGTAGTTCCCAAAAC 3′ (seq idno 4)      XbaI

[0030] was performed to prepare the 3′ flanking sequence. The PCRfragment was cloned as a XhoI/XbaI fragment into XhoI/XbaI cutpCITE/RHDab. The resulting plasmid was called pCITE/RHDabcd.

[0031] Synthetic pox virus promoters were then produced by thehybridization of the following oligonucleotides: Vp3: 5′CTTTTTTTTTTTTTTTTTTTTAGATCTTAAATGCC 3′ (seq id no 5) Vp4: 5′CATGGGCATTTAAGATCTAAAAAAAAAAAAAAAAAAAAGGTA 3′ (seq id no 6)

[0032] These two complementary oligonucleotides anneal together to givecohesive ends compatible with KpnI and NcoI restriction sites. Likewisethe following two oligonucleotides also anneal together give a KpnI/NcoIcompatible fragment. Vp5: 5′ CAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAC3′ (seq id no 7) Vp6: 5′CATGGTATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTTGGTAC 3′ (seq id no 8)

[0033] Vp5 and Vp6 together constitute an early/late promoter, whereasVp3 and Vp4 produce a late promoter (Chakrabarti et al. Biotechniques23, p. 1094-1097, 1997). One or other of the annealed oligonucleotidepairs was then cloned into KpnI/NcoI cut pCITE/RHDabcd, to producepVP/RHD (late promoter) or pEL/RHD (early late promoter). Because theRHD capsid gene is not in frame with the first methionine in either ofthe two constructs (pVP/RHD and pEL/RHD) it was necessary to re-clonethe RHD capsid gene(Vp60) and remove the intervening sequence in orderto obtain expression. The plasmids pVP/RHD and pEL/RHD were cut withNcoI and EcoRI to remove the Vp60 coding sequence and the non codingsequence 5′ to the initiating ATG. The Vp60 gene was then replaced by aPCR generated fragment produced using the following oligonucleotides: 5′GCTCCATGGAGGGCAAAGCCCGTG 3′ (seq id no 9)        NcoI 5′TTGCTCAGGACACCGGCACCTGC 3′ (seq id no 10)

[0034] Template for the PCR reaction was pCITE/RHD. The PCR generatedfragment was digested with NcoI and EcoRI and cloned into the preparedpVP/RHD and pEL/RHD. The resultant plasmids were termed pVP/Vp60 andpEL/Vp60. In order to allow for the introduction of genes flanked byother restriction sites a multiple cloning site was introduceddownstream of the unique NcoI site in pVP/Vp60 and pEL/Vp60. Thefollowing oligonucleotides: mcs A:5′CATGGATCGATGTCGACGGATCCACTAGTGAATTCACGCGTC 3′ (seq id no 11) mcs B:5′TCGAGACGCGTGAATTCACTAGTGGATCCGTCGACATCGATC 3′ (seq id no 12)

[0035] anneal to give overhanging ends compatible with NcoI and XhoIrestriction endonuclease sites. Replacement of the NcoI/XhoI fragment,which carries all of the RHD associated sequence, with the annealedoligonucleoudes results in the following two plasmids: pV_(L) andpV_(EL) (see FIG. 1).

[0036] The plasmids pV_(L) and pV_(EL) were used to construct variousrecombinant DNA plasmids comprising the follwing genes: rabbithaemorrhagic disease capsid protein VP60 (Meyers et al. 1991, supra),green fluorescent protein (Clonetech Laboratories Inc, Palo Alto,Calif., USA), feline leukaemia envelope glycoprotein gp85 (Stewart etal., 1986, J. Virol. 58, p. 825-834), feline leukaemia matrix (gag)proteins (Donahue et al., 1988, J. Virol. 62, p. 722-731), felinecalicivirus capsid protein (GenBank Accession No's Z11536 and NC001481), feline panleukopenia virus capsid protein VP2 (Carlson J. etal. 1985, J. Virol. 55, p. 574-582), canine parvovirus capsid proteinVP2 (Reed P. et al, 1988, J. Virol. 62, p. 266-276). Table 1 lists thevarious DNA plasmids that were constructed to produce the recombinantmyxoma viruses according to the invention. Each recombinant plasmid wasconstructed in the same way, in that the target gene was either cut froman existing plasmid or amplified by PCR such that restriction enzymesites at the ends of the gene were compatible with sites in the mCs ofpV_(L) or pV_(EL).

Preparation of DNA plasmid pV_(L)GFP

[0037] A plasmid containing GFP gene was purchased from Clonetechlaboratories, Inc, Palo Alto, Calif., USA and digested with NcoI andEcoRI to cut the GFP gene from the plasmid. The gene was inserted inpV_(L) resulting in pV_(L)GFP.

Preparation of DNA plasmid pV_(EL)FeLV_(enV)

[0038] The envelope gene was amplified by PCR from pFGA5 (Stewart et al1986 supra) using the oligonucleotides: (seq id no 13) 5′ CAC ATC GATTGA TGG AAA GTC CAA CGC 3′         ClaI (seq id no 14) 5′ TGG AAT TCATGG TCG GTC CGG ATC GTA 3′        EcoRI

[0039] The PCR fragment was digested with ClaI and EcoRI and insertedinto pV_(EL) resulting in pV_(EL)FeLV_(env).

Preparation of DNA Plasmid pV_(EL)FPL

[0040] The FPL capsid gene was obtained by PCR amplification fromreplicative form (rf) DNA by PCR using the following primers: 5′CACATCGATTGATGAGTGATGGAGCAG 3′ (seq id no 15) 5′CGGGAATTCTAGGTGCTAGTTGATATG 3′ (seq id no 16)

[0041] The rf DNA was prepared from feline kidney cells (CRFK) infectedwith a vaccine strain of FPL by standard methods (Reed, P. et al. 1988,J. Virol. 62, p. 266-276)

Preparation of DNA Plasmid pV_(EL)FCV

[0042] Feline kidney cells (CRFK) were infected with FCV strain F9 at amultiplicity of 0.1 pfu per cell. After thirty six hours the cells wereharvested and total RNA was prepared using Guanidine isothiocyanate(TRIzol reagent GibCoBRL). First strand cDNA synthesis was carried outusing oligodt primers (SuperScript Choice GibCo BRL). The completenucleotide sequence and genomic organisation of the F9 strain of FCV hasbeen reported (GenBank Accession Number M86379). Oligonucleotides weresynthesised to prime second strand DNA synthesis and to subsequently PCRamplify the capsid gene. The following oligonucleotides were used toproduce the Vp60 capsid protein gene of FCV (F9 strain): 5′GGATCGATGCGCGGATGACGGGTCAATC 3′ (seq id no 17)     ClaI 5′GGGGACTAGTATTCATAACTTAGTCATGGG 3′ (seq id no 18)         SpeI

[0043] The Vp6ocapsid gene was inserted into the mcs of pV_(L) andpV_(EL) resulting in pV_(L)FCV and pV_(EL)FCV respectively.

Preparation of DNA Plasmid pV_(EL)CPV

[0044] The gene encoding capsid protein VP2 was PCR amplified from CPVvaccine strain of Nobivac Parvo®, digested with NcoI and EcoRI andinserted in pV_(EL).

Example 2 Preparation of Recombinant Myxoma Virus

[0045] A non-pathogenic strain of myxoma virus (designated MR24), whichhad been attenuated by prolonged passage in rabbit kidney cells (RK13)was selected. This attenuated myxoma virus (MR24) was shown to benon-pathogenic (0% mortality) in rabbits when administered to rabbits bythe subcutaneous, intra-dermal or intramusculair routes. MR24 is acandidate myxomatosis vaccine strain for use in rabbits. All viraltitrations and amplifications were carried out in rabbit kidney (RK-13)cells.

[0046] Recombinant myxoma viruses were produced following the methodsdescribed for constructing recombinant vaccinia viruses (Mackett et. al.1985, supra). To do this rabbit kidney cells (RK13) were infected withmyxoma virus MR24 at a multiplicity of 0.1 pfu per cell. After two hoursthe cells were then transfected with plasmid DNA using the lipofectaminetransfection reagent (GibCo BRL). Selection for recombinant viruses wasbased on limiting dilution and identification by immunofluorescence.

[0047] Seventy two hours post transfection the infected/transfected cellcultures were freeze thawed three times in order to release virus. Thisprimary mix of wild type and recombinant virus was diluted 50 fold withtissue culture medium and then 10 microlitres of the virus mix was usedto infect each well of a 96 well tissue culture plate previously seededwith RK13 cells. The 96 well plate was then incubated for 72 hours toallow infection and propagation of the virus to proceed. After this timethe plate was treated to three cycles of freezing and thawing whilstmaintaining the individual status of each well of the plate. This becamethe first round master plate. Subsequently 5 microlitres of viruscontaining medium from each well was plated onto a duplicate 96 wellplate seeded with RK13 cells. After 48 hours the duplicate plate wasfixed with ice cold methanol and the plate screened for expression ofthe recombinant protein by immunofluorescence.

[0048] For example, the cells infected and transfected withpV_(EL)FeLV_(env) were screened for the production of FeLV envelopeprotein as follows; mouse monoclonal antibody 3-17 (European VeterinaryLaboratory, Woerden The Netherlands) ascitic fluid was diluted 1000 foldthen added to each well of the fixed 96 well plate. The plate wasincubated at 37° C. for one hour. The plate was subsequently washed 5times with PBS and then incubated with FITC labeled rabbit anti mouseIgG (Sigma Chemical Co), incubation was then continued for another hour.Finally the plate was washed 5 times with PBS and examined under afluorescence microscope. Wells containing fluorescent foci of infectionwere identified and noted. The corresponding wells from the first roundmaster plate were then diluted over more RK-13 seeded 96 well plates,which in turn became second round master plates. The process of gradualenrichment was continued until recombinant viruses constituted 20-50% ofthe total virus. Expression of the recombinant protein of the otherrecombinant myxoma viruses was screened in a similar way. TABLE 1 DNAplasmids and the corresponding recombinant myxoma viruses. PlasmidRecombinant Virus Strain number pV_(L)/GFP Myxo/GFP Not assignedpV_(L)/VP60 Myxo/RHD Not assigned pV_(EL)/FeLV_(env) Myxo/FeLV_(env)MS0011 PV_(EL)/FCV Myxo/FCV MS0013 PV_(L)/FCV Myxo/FCV MS0014PV_(EL)/FPL Myxo/FPL MS0015 PV_(EL)/CPV Myxo/CPV MS0016

[0049] Final purification of the recombinants was achieved by pickingindividual foci of infection from agar overlaid cultures.

Example 3 myxo/RHD in Chickens

[0050] To determine whether non-lepori species infected with therecombinant myxoma viruses would elicit an antibody response, chickenswere immunized with myxo/RHD by the subcutaneous or intramuscular route.The birds received 10⁵ pfu of virus on day 0 and day 14 of theimmunization schedule. Blood samples were taken at days, 0, 14 and 28and analyzed for antibodies to RHDV, the results are shown in table 2.All the birds remained clinically normal throughout the experiment.TABLE 2 Results of inoculation of chickens with myxo/RHD. Antibodylevels are expressed as a reciprocal of that dilution of sera whichinhibits the agglutination of rabbit red blood cells by 4 units ofpurified RHDV antigen Route of Animal Haemagglutination Inhibition titreInoculation Number Day 0 Day 14 Day 28 Intra-muscular 166 0 80 40 168 040 20 170 0  640-1280 640-1280 172 0 40 40 174 0 320 320 176 0 40 160178 0 320 640 180 0 40 40 sub-cutaneous 182 0 320 320 184 0 40 20 186 010 10 189 0 320-640 320 191 0 40 40 193 0 20 20 195 0 640 640 197 0 320320 Controls 290 0 0 0 292 0 0 0 294 0 0 0 296 0 0 0 298 0 0 0

Example 4 Myxo FCV in Cats

[0051] An experiment was set up to establish the efficacy of amyxoma/feline calicivirus capsid recombinant virus (myxo/FCV) to inducea neutralising antibody response and protect cats from challenge withvirulent feline calicivirus. A group of 4 cats (Group 1) were immunisedsubcutaneously with 5×10⁶ focus forming units (ffu) of myxo/FCV. Theimmunisation was repeated at 3 weeks post first immunisation. Fourunvaccinated control animals (Group 2) were housed with the testanimals. Four weeks after the second immunisation all the cats werechallenged intranasally with 10^(5.3) TCID₅₀ of a virulent strain offeline calicivirus. The challenge virus was introduced dropwise, 0.5 mlinto each nostril. TABLE 3 Schedule of Procedures Time points ANIMALGROUPS PROCEDURE Day - 1 1&2 Swab O/P, nasal. Bleed Day 0 1 VaccinateDay 21 1 Bleed and 2^(nd) vaccination Day 49 1&2 Bleed and ChallengeDays 50-62 1&2 Clinical monitoring Swab O/P, nasal Day 63 1&2 Clinicalmonitoring Bleed Swab O/P, nasal

[0052] Swabs were taken at the start of the experiment to ensure thatnone of the animals had feline calicivirus present in the oro-pharynx.Similarly blood samples were taken to ensure that none of the animalshad anti-FCV antibodies prior to the commencement of the study. Swabswere taken after challenge to examine excretion of virus.

[0053] Blood samples taken during the experiment were used in virusneutralization assays. These assays determine the levels of circulatingantibodies in the cat. It is well known that the serum neutralizingantibodies are present in convalescent animals, and that pre-existentneutralizing antibodies provided as a result of vaccination help inproviding protection from disease (Hohdatsu, et. al. 1999 J. Vet. Med.Sci. 61, 299-301). TABLE 4 Results of inoculation (serum neutralizationtitre) of cats with myxo/FCV (MS0013). The FIGS. show the maximum serumdilution at which virus neutralisation is obtained. The blood samplestaken prior to vaccination (Prebleed) show no antibodies to FCV. Serumdiluted 1-4 fold will show a non-specific inhibition of viral growth invitro. POST POST SERUM Pre- 1^(ST) 2^(ND) Post Clinical Cat No Groupbleed VAC. VAC Challenge Score* 358-054 Vaccinates <1:4  1:102  1:258010321 1 376-561 (Group 1) <1:4  1:50  1:1024 >16384 13 383-882 <1:4 1:215  1:3444 13777 0 073-609 <1:4  1:161  1:1569 16384 4 295-099Controls <1:4 <1:4 <1:5 1337 49 375-369 (Group 2) <1:4 <1:4 <1:4 4096 18078-001 <1:4 <1:4 <1:4 2435 47 268-054 <1:4 <1:4 <1:4 697 22

[0054] Comparing the results obtained after vaccination with myxo-FCV(Table 4) with those of conventional vaccines (Table 5), it is clearthat after the first vaccination the Group 1 animals have an antibodyresponse comparable to animals given two doses of many commercialvaccine. Indicating that these cats would be protected from disease.After the second vaccination the antibody titres are well in excess ofthose obtained by commercial vaccines preparations (Hohdatsu, et. al.1999 J. Vet. Med. Sci. 61, 299-301, DeSilver et. al. 1997, Proc. 1^(st)Int. Symp. Calicivirus ESVV 131-143). TABLE 5 Neutralising antibodytitres of commercially available FCV vaccine immune sera against FCVstrains* Immune serum FCV Vaccine A Vaccine B Vaccine C Vaccine Disolate #20^(a)) #C3 #C1 #C4 #C2 #C5 #C6 #A7 F4  20^(b)) 80 10 5 5 5 16020 F9 160 160 640 40 10 10 160 320 255  10 20 20 5 <5 <5 640 >640 91-1 <5 <5 <5 <5 <5 <5 5 <5

[0055] Feline herpes virus vectors expressing FCV antigens also inducevery low titres of serum neutralising antibodies (in the region 2.5 and3.0) prior to challenge as reported by Yokoyama et. al. (1998).

References

[0056] Hohdatsu T, Sato K, Tajima T & Koyama H. Neutralizing feature ofa commercially available feline calicivirus (FCV) vaccine immune seraagainst FCV field isolates. J Vet Med Sci 1999; 61:299-301.

[0057] Yokoyama N. Fujita K, Damiani et al. Further development of arecombinant feline herepesvirus type 1 vector expressing felinecalicivirus immunogenic antigen. J of Vet Med Sci 1998; 60:717-723.

[0058] Reed L J & Meunch H. A simple method of estimating fifty per centend points. Am J of Hygiene 1938; 27:493-497.

Example 5 Netralization Experiments with Myxo/FCV in Pigs and Bovines

[0059] The applicability of the myxoma virus as an eukaryotic expressionvector for the induction of a protective immune response in non-naturalhosts i.e. bovine and pigs was tested by intra-dermal and intra-muscularroute of injection.

[0060] A myxoma virus vector containing the feline calicivirus genefragment encoding for the mature form of the capsid antigen was usedboth in calves as well as pigs. Two groups comprising four calves of 12weeks old and four pigs of 6 weeks old were included in this study.

[0061] Blood samples (10 ml/animal) were collected one week before startof the study in order to test whether the animals were sero-negative tofeline calicivirus.

[0062] Subsequently, all animals were injected both intra-dermally aswell as intramuscularly with 1 ml PBS containing 1×10⁸ FFU of therecombinant myxoma virus/route of injection/animal. The animals werehoused as one group per species. After four weeks, blood samples (10ml/animal) were collected and animals were re-injected intra-dermallyand intramuscularly with the same dose of myxoma virus i.e. 1 ml of1×10⁸ FFU of the recombinant myxoma virus/route of injection/animal. Twoweeks after the last injection, blood samples (10 ml/animal) werecollected and animals were destroyed according to the guidelines for GMOtesting in vivo. Collected blood samples were tested for the presence ofantibodies directed against Calicivirus by means of a virusneutralisation assay in vitro. TABLE 6 Experimental design and timeframe: Groups consist either of four pigs or four calves. Date ActivityT = −7 days Pre-serum samples were collected T = 0 days Intra-dermal andintra-muscular injection with 1 ml PBS containing 1 × 10⁸ FFU of myxomavirus per route of injection and per animal T = 28 days p.i. Bloodsamples were collected (10 ml) T = 28 days p.i. Intra-dermal andintra-muscular injection with 1 ml PBS containing 1 × 10⁸ FFU of myxomavirus per route of injection and per animal T = 42 days p.i. Bloodsamples were collected T = 42 days p.i. Termination of experiment

[0063] Method For FCV serum neutralisation assay

[0064] Serum neutralisation was assessed by c.p.e. on CrFK cells. Fivefold replicates of two hundred TCID₅₀ of virus were mixed with serialdilutions, (commencing at 1:4), bf sera in a final volume of 600microliter. Virus/sera mixtures were then incubated for 60 minutes at37° C. in sterile 5 ml dilution tubes. 100 microliter of the testmixtures were then added 96 well tissue culture dishes seeded with CrFKcells in 100 microliter growth medium. Incubation was continued for 5days. The TCID₅₀ values were calculated according to the method of Reed& Meunch [1].

[0065] Reed, L. J., and H. Meunch. 1938. A simple method of estimatingfifty percent end points. Am. J. Hyg., 27:493-497. TABLE 7 Results ofSns against FCV on Sera from Study FMD004 Animal Prebleed Post 1^(st)Vac Post 2^(nd) Vac Bovine 2403 <=1:4 1:54 >=1:724 Bovine 1948 <=1:41:64  >=1:1024 Bovine 1603 <=1:4 1:13     1:256 Bovine1190 <=1:41:54 >=1:861 Pig 122 <=1:4 <=1:4          1:304 Pig 119 <=1:4 <=1:4         1:304 Pig 118 <=1:4 1:10     1:256 Pig 117 <=1:4 <=1:4      >=1:861

Example 6 Growth of Myxoma Virus Strains in Various Cell Types in Vitro

[0066] Cells were infected with recombinant myxoma viruses expressinggreen fluorescence protein constructed from 2 different strains ofmyxoma virus. Growth was assayed by an increase in the number offluorescing cells over time

[0067] Fluorescence was observed when the myxo/GFP recombinant virus wasplated onto various cell types in culture, as shown in table 8. Thisindicated that the virus was able to enter and express the GFP gene innon rabbit cells. From Table 8 it shows that growth of the virus invitro was observed in some cell types, and that the pattern differed forthe two constructs tested. TABLE 8 growth of myxoma vectors in variouscell types in vitro. Growth Cell Type MS20-10 MR24 Rabbit Kidney ++++++++++ (RK-13) African monkey +++++ +++++ kidney (Vero) Bovine embryo + +lung (BEL) Feline embyo + − fibroblasts (FeF) Chicken embryo + −fibroblasts (CEF) Canine Tumor cell line A72 − −

Example 8 ELISA For Responses to Canine Parvovirus Vaccination

[0068] In order to assess the immune responses elicited by vaccinationof dogs with a recombinant myxoma virus expressing canine parvoviruscapsid protein Vp2 an ELISA was setup. The responses to vaccination werecompared with those found in conventionally vaccinated dogs

[0069] Materials & Methods

[0070] Mateials:

[0071] TBS (50 mM Tris buffered saline)

[0072] 50 mM Tris-buffered Saline, pH 7.5

[0073] 6.35 g Tris-HCl

[0074] 1.18 g Tris Base

[0075] 8.77 g NaCl

[0076] 800 ml dH₂O

[0077] pH adjusted to 7.5 and volume brought to 1 L with dH₂O.

[0078] TBS-Tween

[0079] TBS was prepared as above then add 0.5 mL of TWEEN 20. Mix Well

[0080] Methods

[0081] 1. Anti CPV monoclonal antibody was resuspended in 0.1 M Na₂ CO₃buffer pH9.6 at a concentration of 5-10 microgram/ml. An ELISA plate wasincubated overnight at 40C. with 100 microliter per well of the antibodysuspension.

[0082] 2. After shaking off excess antigen coating solution, remainingbinding sites were blocked in each well by incubating with 200microliter of 1% BSA and 2% dry milk powder in TBS at room temperaturefor one hour.

[0083] 3. After shaking off the block solution, it was replaced with 100microliter of tissue culture supernatant containing canine parvovirus ata titre of approx. 10⁷ p.f.u. ml⁻¹. Incubation was carried out at roomtemperature for 1-2 hours.

[0084] 4. Plates were washed four times with TBS-Tween.

[0085] 5. Serial dilutions of the serum to be tested were made in TBS.100 microliter of these were added to the wells of the ELISA plate andincubation was continued for 1-2 hours at room temperature.

[0086] 6. Afterwards the plate was washed four times in TBS-Tween.

[0087] 7. An anti-dog alkaline phosphatase conjugated second antibody,(e.g ICN biomedical Research Products cat no. 675071) was added at adilution indicated by the manufacturer. Incubation was carried out atroom temperature for 1-2 hours

[0088] 8. The plate was washed four times in TBS-Tween

[0089] 9. The ELISA was developed by the addition of substrate PNPP(p-Nitrophenyl phosphate e.g SIGMA chemical company cat number N2770).

[0090] 10. Absorbance was read in a spectrophotometer at 420 nm.

[0091] Results are presented in Table 9. TABLE 9 results of ELISA forresponses to CPV Absorbance at indicated dilution Vaccine 10 20 40 80160 320 640 1280 Myxo-Vp2 >2.0 >2.0 1.8 1.56 1.198 0.685 0.45 0.297Conventional 0.73 0.46 0.29 0.30 0.28 0.30 0.26 0.29 None 0.31 0.28 0.300.26 0.28 0.25 0.27 0.26

[0092] The ELISA results clearly demonstrate that a dilution of 1:40 ofthe sera from conventionally vaccinated dogs results in a backgroundlevel of aborbance i.e. that seen with unvaccinated dog sera. Whereaswith myxoma-CPV(Vp2) vaccinated dog sera a dilution of 1:1280 isrequired to achieve the same background level.

1. Use of a live, recombinant leporipox virus comprising exogenous DNA,which is operably linked to at least one expression expression controlelement and which is incorporated in a non-essential region of the virusgenome, in the manufacture of a vector vaccine for the treatment and/orprophylaxis of infectious diseases in non-lepori species.
 2. Use of avirus according to claim 1 in the manufacture of a vector vaccine forthe treatment and/or prophylaxis of infectious diseases in felines orcanines.
 3. Vaccine comprising a pharmaceutical acceptable carrier and alive recombinant leporipox virus comprising exogenous DNA operablylinked to at least one expression control element and incorporated in anon-essential region of the virus genome, said exogenous DNA encoding atleast one antigen of a pathogen that produces an infectious disease innon-leporidae.
 4. A live, recombinant leporipox virus comprisingexogenous DNA operably linked to at least one expression control elementand incorporated in a non-essential region of the virus genomecharacterized in that said exogenous DNA encodes at least one antigen ofa non-lepori pathogen.
 5. A virus according to claim 4 characterized inthat the leporipox virus is a myxoma virus.
 6. A virus according toclaim 4 or 5 characterized in that the exogenous DNA encodes at least anantigen of a feline or canine pathogen.
 7. A virus according to claims 4or 5 characterized in that the exogenous DNA encodes at least an antigenof a feline or canine virus.
 8. A virus according to claims 4 to 7characterized in that the exogenous DNA encodes Feline Leukaemia virus(FeLV) the envelope protein, the Feline Calicivirus (FCV) capsidprotein, the Feline Panleukopenia virus (FPL) VP2 protein, and/or CanineParvovirus (CPV) VP2.
 9. A virus according to claim 4 to 8 characterizedin that the exogenous DNA and expression control element are inserted inthe MGF ORF of the virus genome.
 10. A virus according to claim 4 to 9characterized in that the expression control element operably linked tothe exogenous DNA is a synthetic poxvirus promoter.
 11. A virusaccording to claim 10 characterized in that the promoter is anearly/late promoter.